Although much is known about exocytosis in chromaffin cells, little is known about secretory granule movement close to the plasma membrane preceding the final fusion event. A key aspect of the proposal is our ability to visualize chromaffin granules in real time. A fusion protein of atrial natriuretic peptide and green fluorescent protein that is expressed by transient transfection is sorted to chromaffin granules. Using the optical technique of total internal reflection, we can visualize fluorescent chromaffin granules in living cells that are localized within 1-2 granule diameters of the plasma membrane. I hypothesize that movement and placement of chromaffin granules close to the plasma membrane are highly regulated processes that help define the availability of granules to undergo exocytosis. The focus of this proposal is to determine whether cofactors and proteins known to influence the final secretory response act at least in part by controlling these prefusion events. My specific aims are the following: (1) to determine the location and movement of chromaffin granules close to the plasma membrane under basal and secretory conditions, (2) to determine the relationship between the known effects of ATP and Ca2+ on granule priming before secretion and granule movement adjacent to the plasma membrane and (3) to determine the role of SNAREs and Rab3a, specific proteins of the secretory pathway in granule movement and placement adjacent to the plasma membrane.